On addition of NADPH to rat liver microsomes cytochrome b5 assumes a steady-state reduction level, based on a balance between its reduction by NADPH and its aut oxidation. Initiation of fatty acid desaturation by adding stearoyl-CoA(stearyl CoA) results in a rapid shift of the NADPH-supported steady state of the cytochrome in favor cf more oxidation, and this shift is intensified by partially inhibiting microsomal NADPH-specific flavoprotein by HgCl2 This suggests that increased utilization of reducing equivalents for desaturation is accompanied by a stimulation of reoxidation of cvtochrome b5. Actually, the magnitude of stearyl CoA-induced shift is roughly proportional to the fatty acid desaturation activity of the microsomes employed. Moreover, this shift is interfered with by cyanide which inhibits a terminal component (" cyanide-sensitive factor") of the desaturation system. Since the reduction of microsomal bound cytochrome b5 by NADH is much faster than the autoxidation of the cytochrome and the overall desaturation reaction, NADH causes complete reduction of cytochrome b5 and no change in the reduction level is observed on addition of stearyl CoA. However, when NADH-cytochrome b5 reductase is strongly inhibited by PCMS, stearyl CoA does lower the steady-state reduction level of cytochrome b5 in NADH-treated microsomes. Stearyl CoA also stimulates the oxidation of cytochrome b5 observable on exhaustion of the NADH added, and this stimulation is again prevented by cyanide. With microsomes from which cytochrome b5 has been removed to various extents by mild proteolytic digestion, the desaturation of stearyl CoA supported by ascorbate as electron donor is dependent on the content of remaining cytochrome b5. It is concluded that cytochrome b5 in liver microsomes acts as an intermediary electron carrier which passes reducing equivalents from NADH, NADPH and ascorbate to the cyanide-sensitive factor where fatty acid desaturation per se probably takes place. Micresomes prepared from rat epididymal adipose tissue contain a significant amount of cytochrome h, which plays a similar role in fatty acid desaturation.